Urea Lysis Buffer System
Urea Lysis Buffer Materials Buffers containing Urea cannot be sterilized so ensure all materials are sterileprior to preparation of buffers. Buffers are stable for at least 6 months at room temperature. Do not store Urea Lysis Buffer or Wash Buffers at temperatures lower than room temperature (25 C) as the Urea will come out of solution by crystallization and temperatures higher than room temperature as the urea will start to degrade and form pthalates. *Monosodium phosphate, NaH2PO4 (1 M) *Disodium phosphate, Na2HPO4 (1 M) *Urea (crystal) *Tris-Cl (1 M, pH 8) *Imidazole (1 M) Method (makes 1 L of Lysis buffer) Urea Lysis Buffer contains 5 mM imidazole to reduce binding of non-tagged proteins. pH is high and is subsequently lowered as resin is washed. #In a liter bottle, add 6.8 mL of Monosodium phosphate. #Add 93.2 mL of Disodium phosphate to the bottle. #Add 10 mL of Tris-Cl (pH 8). #Add 10 mL of Imidazole solution. #Add 480.48 grams of Urea and bing up the volume to 100 mL with deionized distilled water. Dissolve by stirring. #Store at room temperature. Wash Buffer B Materials *Monosodium phosphate, NaH2PO4 (1 M) *Disodium phosphate, Na2HPO4 (1 M) *Urea (crystal) *Tris-Cl (1 M, pH 6.4) *Imidazole (1 M) *Triton X-100 (100%) Method (makes 1 L of Wash buffer) pH of Wash Buffer B is lower and also contains 0.2% Triton- X-100 detergent. #In a liter bottle, add 74.5 mL of Monosodium phosphate. #Add 25.5 mL of Disodium phosphate to the bottle. #Add 10 mL of Tris-Cl (pH 6.4). #Add 10 mL of Imidazole solution. #Add 2 mL of Triton X-100 detergent. #Add 480.48 grams of Urea and bing up the volume to 100 mL with deionized distilled water. Dissolve by stirring. #Store at room temperature. Wash Buffer C Materials *Monosodium phosphate, NaH2PO4 (1 M) *Disodium phosphate, Na2HPO4 (1 M) *Urea (crystal) *Tris-Cl (1 M, pH 6.2) *Imidazole (1 M) *Triton X-100 (100%) Method (makes 1 L of Wash buffer) pH of Wash Buffer C is still lower and contains 0.1% Triton- X-100 detergent. #In a liter bottle, add 82.2 mL of Monosodium phosphate. #Add 17.8 mL of Disodium phosphate to the bottle. #Add 10 mL of Tris-Cl (pH 6.4). #Add 10 mL of Imidazole solution. #Add 1 mL of Triton X-100 detergent. #Add 480.48 grams of Urea and bing up the volume to 100 mL with deionized distilled water. Dissolve by stirring. #Store at room temperature. TBS Elution Buffer Materials *Tris-Cl (1 M, pH 7.5) *NaCl (1 M) *Imidazole (1 M) Method (makes 100 mL of buffer) This elution buffer is a TBS-based buffer with imidazole for the elution of His-tagged proteins from Ni-NTA resin. TBS+imidazole elution buffer is stable at 4 C for 3 months. #Add 5 mL of Tris-Cl to a bottle. #Add 15 mL of NaCl to the bottle. #Add 25 mL of Imidazole solution and then bring up the volume to 100 mL with deionized distilled water. #Sterilize by autoclaving and store at 4 C (fridge). Category:Urea Category:Lysis Category:Denaturing Category:Affinity Purification